Methods for treating HIV-Infected Patients by the Administration of GM-CSF and a protease inhibitor

ABSTRACT

Provided are methods for inducing an increase in the number of CD4 +  T-lymphocytes in HIV-infected patients by administering human GM-CSF. The GM-CSF may be administered concurrently with at least one antiretroviral agent.

FIELD OF THE INVENTION

The subject invention relates to methods for increasing the number of circulating CD4⁺ T-lymphocytes in HIV-infected patients by the administration of granulocyte-macrophage colony stimulating factor (GM-CSF).

BACKGROUND OF THE INVENTION

Patients infected with Human Immunodeficiency Virus (HIV) experience a variable but progressive decline in immune function resulting in clinically apparent opportunistic infections and other diseases. (Crowe et al., J. Acquir. Immune Defic. Syndr. 4:770-76, 1991; Moss et al., AIDS 3:55-61, 1989). The onset of severe immunodeficiency in HIV-infected individuals is generally accompanied by a marked increase in viral load and a dramatic decline in circulating CD4⁺ T-lymphocytes. Indeed, one of the clinical criteria for the diagnosis and reporting of AIDS (as established by the Centers for Disease Control and Prevention) is a decrease in the number of CD4⁺ T-lymphocytes to <200 cells/mL. (Normal CD4⁺ T-lymphocyte cell counts in healthy HIV⁻ individuals range between 800 and 1600 cells/mL.) CD4⁺ T-lymphocytes perform multiple immune-modulating functions. The decline in CD4⁺ T-lymphocytes, and decline in cell-mediated immunity, is the primary factor responsible for the susceptibility of HIV-infected patients to many opportunistic infections characteristic of the adult immunodeficiency syndrome, or AIDS. Accordingly, an increase in CD4⁺ T-lymphocytes is generally regarded as an indicator of efficacy for anti-HIV drugs.

Inhibition of HIV replication can increase CD4⁺ T-lymphocyte counts. Such antiretroviral therapy typically involves combinations of drugs such as protease inhibitors, nucleoside analogs, and non-nucleoside reverse transcriptase inhibitors. Other agents, including biologics, have also demonstrated some antiviral effects. The decrease in viral load is generally, but not always, associated with an increase in the number of circulating CD4⁺ T-cells. (Yarchoan et al., Ann Intern. Med. 115:184-89, 1991; Hirsch and D'Aquila, N. Engl. J. Med. 328:1686-95, 1993; Volberding, P. A., In: Crowe et al., eds., Management of the HIV-Infected Patient, pp. 53-63). Unfortunately, antiretroviral drugs do not result in complete reconstitution of the immune function. Moreover, inhibition of viral replication by these agents is temporary, due to the evolution of resistant strains of virus that can grow in the presence of the antiretroviral agents. (Cameroni et al., Third Human Retroviral Conference, January 1996, Abstract #LB6a).

Growth factor cytokines such as granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (EPO) have also been administered to patients with HIV. (Scadden et al., 1991, Levine et al., Blood 73:3148-54, 1991; Kaplan et al., J. Clin. Oncol. 9:929-40, 1991; Stricken and Goldberg, Clin. Immunol. Immunopathol. 79:194-96, 1996; Miles et al., Blood 77:2109-17, 1991; Pluda et al., Hematol. Oncol. Clin. North Am. 5:229-48, 1991). Recently, GM-CSF has been the subject of several studies to evaluate its ability to prevent opportunistic infections in individuals with HIV.

LEUKINE®, a yeast-derived form of GM-CSF, is currently available for use in promoting myeloid cell recovery following bone marrow transplant post-myeloablative therapy for the treatment of malignancies. An E. coli-derived form of GM-CSF is also available for use in promoting the recovery of neutrophils in HIV-infected patients with granulocytopenia. (Scadden et al., J. Clin. Oncol. 9:802-08, 1991; Levine et al., Blood 78:3148-54, 1991; Kaplan et al., J. Clin. Oncol. 9:929-40, 1991; Hardy et al., Eur. J. Clin. Microbiol. Infect. Dis. 13:S34-S40, 1994). However, the widespread use of GM-CSF for treatment of HIV infection has been hindered by data from in vitro studies whose results suggest that this cytokine might actually enhance HIV replication (Bender et al., J. Immunol. 151:5416, 1993; Foli et al., Blood 8:2114, 1995; Pluda et al., Hematol. Oncol. Clin. North Am. 5:229-48, 1991). More recent studies have reported results which are inconsistent with earlier studies with regard to the effect of GM-CSF on HIV viral replication (Perno et al., Third Human Retroviral Conf., January 1996, Abstract #463; Pluda et al., Blood 76:463-72, 1990; Fletcher and Gasson, Blood 71:652-58, 1988; Mitsuyasu, R. T. In: Volberding and Jacobson, eds. AIDS Clinical Review, N.Y., N.Y. 1993-94, pp. 189-212). It is now believed that in the presence of antiretroviral therapy, GM-CSF does not upregulate HIV viral replication. (Scadden et al., 1995; Davison et al., J. Clin. Pathol. 47:855-57, 1994, Scadden et al., AIDS Res. and Human Retroviruses 12:1151-59, 1996). Indeed, in vitro data have demonstrated the enhancement of AZT activity by GM-CSF due to increased intracellular concentration of the active triphosphorylated form of AZT. (Hammer and Gillis, Antimicrob. Agents Chemother. 31:1046-50, 1987).

Developing effective therapies for HIV disease has presented a formidable challenge for medical researchers. Although significant advancements have been made in the treatment of HIV-infected patients, many patients remain untreatable due to ineffectiveness of the therapeutic drugs used or inability of the patients to tolerate the side effects of the therapies. Clearly, existing therapies do not yet offer a cure to HIV disease. Immune-modulating agents, such as GM-CSF, may therefore offer an additional alternative treatment.

SUMMARY OF THE INVENTION

The present invention provides methods for increasing CD4⁺ lymphocyte counts in HIV-infected patients by the therapeutic administration of GM-CSF. This method of treatment has been demonstrated to induce an increase in the absolute number of circulating CD4⁺ T-lymphocyte cells in patients concurrently receiving antiretroviral drugs, with no significant increase in viral load.

This invention is based on the results of a double-blinded, placebo-controlled study that enrolled HIV-infected patients at two study sites. In this study, patients receiving anti-retroviral agents for a minimum of eight weeks prior to study received therapeutically effective amounts of recombinant human GM-CSF or placebo. Viral load and CD4⁺ T-lymphocyte counts were determined twice prior to the start of the study (baseline), then two weeks, four weeks, and eight weeks after the start of the study, and twice approximately four weeks after the treatment phase was completed. Results indicated that within the placebo arm of the study, there was no significant change in the median values for CD4⁺ T-cell counts relative to the baseline. Within the GM-CSF arm of the study, a trend towards an increase in the median value for CD4⁺ T-lymphocyte counts was observed between the baseline and all evaluations during the course of the study.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides methods for inducing an increase in the number of CD4⁺ T lymphocytes in an HIV-infected patient by administering therapeutically effective amounts of granulocyte-macrophage stimulating factor (GM-CSF).

In accordance with the present invention, GM-CSF is administered to HIV-infected patients in amounts and for a time sufficient to induce a clinically significant increase in the patient's CD4⁺ T-lymphocyte count. A “CD4⁺ T-lymphocyte count” means the number of circulating CD4⁺ T-lymphocytes in the patient's blood, expressed as CD4⁺ T-lymphocyte cells per unit volume. Generally, CD4⁺ lymphocyte counts are expressed as cells/mm³ of whole blood. An increase in the CD4⁺ T-lymphocyte count is defined as a rise relative to the baseline (i.e., before administration of GM-CSF) value. For the purposes of the present invention, a “clinically significant” increase in CD4⁺ T-lymphocyte cells is an increase of greater than or equal to about 30% of the baseline value.

GM-CSF used in the practice of the invention includes any pharmaceutically safe and effective human GM-CSF, or any derivative thereof having the biological activity of human GM-CSF. In a presently preferred embodiment, the GM-CSF used in the practice of the subject methods is recombinant human GM-CSF (rhu GM-CSF), such as LEUKINE® (Immunex Corporation, Seattle, Wash.). LEUKINE® is a biosynthetic, yeast-derived, recombinant human GM-CSF, consisting of a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF by having a leucine instead of a proline at position 23. Other natural and synthetic GM-CSFs, and derivatives thereof having the biological activity of natural human GM-CSF, will of course be equally useful in the practice of the invention.

As the degree of glycosylation of biosynthetic GM-CSFs appears to influence half-life, distribution, and elimination, the most effective dose of GM-CSF for the subject methods may vary depending on the source used (Lieschke and Burgess, N. Engl. J. Med. 327:28-35, 1992; Dorr, R. T., Clin. Ther. 15:19-29, 1993; Horgaard et al., Eur. J. Hematol. 50:32-36, 1993). The optimal dose of GM-CSF used for LEUKINE® may be adjusted if a GM-CSF other than LEUKINE® is used to induce CD4⁺ T-lymphocyte cells in HIV-infected patients.

LEUKINE® has been shown to exhibit the same hematopoietic effects as those induced by endogenous GM-CSF, namely, the stimulation of progenitor cells committed along the granulocyte-macrophage pathway to form neutrophils, monocytes, macrophages, and eosinophils (Technical Product Report: LEUKINE® Liquid, Immunex Corp., Seattle, Wash., 1997, which is herein incorporated by reference). LEUKINE®, like endogenous GM-CSF, also promotes the differentiation of progenitor cells giving rise to erythrocytes and megakaryocytes (Ibid.) In addition to stimulating hematopoiesis, LEUKINE® enhances many of the functional activities of mature neutrophils, monocytes and macrophages, such as chemotaxis, growth factor secretion, anti-tumor activity, antibacterial and antifungal activities, and so on (Ibid.).

Various embodiments of the subject invention are disclosed herein. In one preferred embodiment, GM-CSF may be administered concurrently with antiretroviral agents, including, but not limited to, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, or protease inhibitors. The term “antiretroviral agent”, as used herein, includes any pharmacological, biological or cellular agent that has demonstrated the ability to inhibit HIV replication. Specific examples of nucleoside reverse transcriptase inhibitors include zidovudine (AZT), didanosine (ddI), lamivudine (3TC), stavudine (d4T), and dalcitabine (ddC). Specific examples of non-nucleoside reverse transcriptase inhibitors include nevirapine and delavirdine. Specific examples of protease inhibitors include indinavir, nelfinavir, ritonavir, and saquinavir. Patients treated in accordance with the present invention may be treated concurrently with one or more anti-retroviral agents. Additional antiretroviral agents not yet approved by the Food and Drug Administration may also be effective.

The optimal dose, frequency of administration, and duration of treatment with GM-CSF which is effective to induce a clinically significant increase in CD4⁺ T-lymphocyte counts may vary from patient to patient. Generally, however, therapeutically effective doses of GM-CSF sufficient to induce an increase in the patient's CD4⁺ T-lymphocyte count will be greater than or equal to about 100 micrograms (mcg). Preferably, doses of GM-CSF will be greater than or equal to about 150 mcg, and more preferably, doses of GM-CSF will be greater than or equal to about 250 mcg.

In preferred embodiments of the present invention, GM-CSF is administered for a period of time greater than about three weeks, and more preferably greater than about four weeks, at a frequency of at least two times per week, more preferably at least three times per week, and most preferably once per day or more. However, it should be understood that the optimal dose and length of treatment may vary from patient to patient, depending on the individual patient's condition and response to the treatment, and is best determined by monitoring the patient's response during the course of the treatment. It should further be understood that administration of higher doses may permit less frequent administration, and lower doses may require more frequent administration in order to achieve clinically significant increases of CD4⁺ T-lymphocyte counts. A treatment regimen (dosage amount, frequency and duration) is therapeutically effective if it results in a clinically significant increase in CD4⁺ T-lymphocyte counts.

The methods of the subject invention thus include inducing an increase in the CD4⁺ T-cell count in an HIV-infected patient who may also be treated with an antiretroviral agent in which the patient is administered an amount of GM-CSF sufficient to induce an increase in the patient's CD4⁺ T-lymphocyte count.

EXAMPLE Study Design

A study was conducted to determine the safety and efficacy of human GM-CSF as a therapeutic reagent in HIV-infected patients. Because some in vitro studies had indicated the possibility that GM-CSF might enhance HIV replication in vivo, this study assessed the impact of co-administration of GM-CSF with an antiretroviral agent on HIV replication. HIV viral burden was measured by the RNA-PCR (AMPLICOR®) method. To assess the effects of this treatment on lymphocyte counts, blood samples were analyzed throughout the study for determination of CD3⁺, CD4⁺, and CD8⁺ T-lymphocyte counts.

This study was a Phase I/II, randomized, double-blind, placebo-controlled, two-center trial involving patients with HIV disease. Patients meeting specific entry criteria were randomized to treatment with GM-CSF or placebo, and these were administered by subcutaneous injection three times per week for eight weeks. Viral load was determined at baseline (mean of three determinations within fourteen days of study entry), then at two weeks, four weeks, and eight weeks after starting study drugs, and on two separate occasions approximately four weeks after the conclusion of the treatment phase (post-treatment evaluation). Clinical and lymphocyte subset evaluations were performed at each of these same time points (i.e., pre-treatment, week 2, week 4, week 8, and 4 weeks post-study). Twenty patients were enrolled in the study, ten per treatment arm. All twenty patients completed the study.

Patients were eligible for inclusion in the study if the following parameters were met: documented HIV infection, including detectable RNA-PCR during baseline evaluation period, age ≧18 years (or legal age of consent), adequate hematologic function, satisfactory hepatic and renal function, adequate clinical performance status, treatment with an antiretroviral regimen that included either ritonavir or indinavir for at least 8 weeks prior to study enrollment and agreement to continue on a stable regimen throughout the study, ability and willingness to provide written informed consent, agreement to practice medically-approved contraception.

Patients were ineligible for inclusion in the study if the following exclusion criteria were met: concurrent pregnancy or breast feeding, concurrent use of other colony stimulating factors or cytokines (other than erythropoietin), life-expectancy <90 days, concurrent radiation or systemic cancer chemotherapy (or within 2 weeks prior to study entry), inability to self-administer or arrange for administration of subcutaneous injections, significant cardiac disease, prior history of adverse reaction to yeast-derived rhu GM-CSF which would prohibit retreatment, active infection (AIDS-related or not) requiring acute systemic therapy within 8 weeks prior to study enrollment, vaccination of any kind within 8 weeks of study enrollment.

Study participants were randomly assigned to receive either active GM-CSF or placebo. Patients were instructed in methods for self-administration of study medication and received preloaded syringes or vials with empty syringes for home use.

The formulation of recombinant human GM-CSF utilized in this clinical trial was LEUKINE® (commercially available from Immunex Corporation, Seattle, Wash.). LEUKINE® and placebo were supplied as lyophilized powders. Both LEUKINE® and placebo were reconstituted by the aseptic injection of Bacteriostatic Water for Injection, USP, containing 0.9% benzyl alcohol. The placebo control for the subject study was a sterile lyophilized preparation containing only the inactive excipients present in the LEUKINE® preparation. These were 40 mg mannitol, USP; 10 mg sucrose, NF; and 1.2 mg TRIS (tromethamine), USP; per vial of study drug. Vials containing placebo were labeled in a fashion identical to vials containing LEUKINE®. Vials of placebo and LEUKINE® were stored refrigerated at 2-8° C. (36-46° F.).

Patients assigned randomly to treatment Group I received blinded subcutaneous injections of active reconstituted lyophilized LEUKINE® in a fixed dose of 250 mcg in a volume of 0.5 mL three times per week for eight weeks (24 doses), while patients assigned to treatment Group II received blinded subcutaneous injections of 0.5 mL of the placebo according to the same schedule. Safety evaluations included monitoring of HIV viral load by sensitive RNA-PCR methods and clinical monitoring for HIV-related illnesses.

At 2, 4 and 8 weeks after the start of study drug and at follow-up (approximately 4 weeks after completion of study drug), patients were examined, their medical histories were updated, and samples were taken for laboratory studies. A central laboratory was utilized for HIV quantification laboratory tests in order to standardize procedures and minimize variability. These samples were batched by subject to further reduce variability and, with exception of the screening RNA-PCR, results were not available to the study site until after the completion of the study. All other laboratory evaluations were analyzed at licensed laboratories near the study site. Samples for these other tests were drawn at screening, baseline (pre-treatment), week 2, week 4, week 8, and 4 weeks post-discontinuation. These other laboratory evaluations included: Complete blood count (CBC) with differential, platelet count, serum chemistries (electrolytes, blood urea and nitrogen (BUN), creatinine, bilirubin, aspartate amino transaminase (AST), alkaline phosphatase), CD3/CD4/CD8 lymphocyte absolute counts and percentage of total lymphocytes. Additionally, plasma was saved for future cytokine analyses. A single urinalysis was done at baseline to document eligibility.

Results of the Study

The results for individual patients are summarized in the following Table 1A (placebo treatment) and Table 1B (GM-CSF treatment). These results are summarized in the following Table 2.

It is apparent that within the placebo arm of the study (Table 1A), the median values for CD4⁺ T-lymphocyte counts changed very little throughout the course of treatment, and by the time of the post-treatment evaluation, were slightly lower than the baseline CD4⁺ T-lymphocyte level. In contrast, the median values for CD4⁺ T-lymphocyte counts for the GM-CSF arm of the study (Table 1B) were increased at all of the evaluation points and overall to a higher degree than in the placebo group. With respect to individual patients, only three (101/RI, 103/RI and 110/RI) of the ten patients in the placebo arm (30%) experienced an increase in CD4⁺ lymphocyte counts greater than or equal to 30% through week 8, while six (104/RI, 107/RI, 107/RI, 109/RI, 111/RI, 204/RI, and 207/RI) of the ten patients (60%) receiving GM-CSF experienced an increase in CD4⁺ lymphocyte counts greater than or equal to 30% through week 8. Thus, twice as many patients in the GM-CSF arm of the study experienced clinically significant increases in CD4⁺ lymphocyte counts as compared to the placebo arm of the study.

The absolute data shown in Table 2 are summarized in Tables 1A and 1B as changes in CD4⁺ lymphocyte counts. In Tables 1A and 1B, “ND” mean tests were not done. In Table 2, “N” is the number of patients for whom CD4⁺ lymphocyte counts were done at each time point, and “n” is the number of patients exhibiting a change greater than or equal to 30%. No significant differences were observed between the GM-CSF group and the placebo group with respect to changes in viral load throughout the course of the study.

Thus, the results of the studies indicate that the administration of GM-CSF results in a significant increase in the overall number of CD4⁺ lymphocyte cells without a concurrent increase in viral load.

TABLE 1A Absolute CD4⁺ T-Lymphocyte Counts Placebo Arm Post- Patient First Baseline Week 2 Week 4 Week 8 Study #/Site Dose CD4 CD4 CD4 CD4 CD4 101/RI Nov. 18, 1996 10 46 35 35 59 103/RI Jan. 23, 1997 47 89 71 108 105 106/RI Feb. 18, 1997 90 92 56 81 50 108/RI Feb. 25, 1997 386 292 328 454 368 110/RI Mar. 2, 1997 231 397 296 317 302 201/NY Sep. 4, 1996 502 602 647 592 541 203/NY Sep. 4, 1996 87 88 71 66 61 206/NY Sep. 19, 1996 437 388 439 402 399 208/NY Jan. 21, 1997 389 428 378 454 374 209/NY Feb. 18, 1997 248 228 259 220 166 Median 240 260 278 269 234

TABLE 1B Absolute CD4⁺ T-Lymphocyte Counts GM-CSF Arm Base- Post- Patient First line Week 2 Week 4 Week 8 Study #/Site Dose CD4 CD4 CD4 CD4 CD4 102/RI Jan. 21, 1997 24 15 26 18 136 104/RI Jan. 27, 1997 136 157 182 176 200 105/RI Feb. 6, 1997 276 229 356 238 208 107/RI Jan. 25, 1997 383 304 477 750 373 109/RI Mar. 3, 1997 65 227 31 76 77 111/RI Mar. 20, 1997 590 979 725 1014 659 202/NY Nov. 4, 1996 152 77 91 84 199 204/NY Nov. 5, 1996 91 ND 219 162 161 205/NY Nov. 7, 1996 24 25 12 21 11 207/NY Dec. 31, 1996 40 26 61 29 33 Median 114 157 137 123 180

TABLE 2 Change in CD⁴⁺ T-Lymphocyte Counts Relative to Baseline PLACEBO GM-CSF WEEK 2 Mean (Sd) 22 (74) 39 (150) Median 19 −9 Min - Max −94-166 −79-389 No. Patients 10 9 WEEK 4 Mean (Sd) 15 (57) 40 (68) Median 7 34 Min - Max −58-145 −61-135 No. Patients 10 10 WEEK 8 Mean (Sd) 30 (50) 79 (172) Median 43 4 Min - Max −35-90 −68-424 No. Patients 10 10 WEEK 12 Mean (Sd) 0 (51) 28 (54) Median −17 30 Min - Max −82-71 −68-112 No. Patients 10 10 MAXIMUM CHANGE > n/N (%) 3/10 (30%) 6/10 (60%) 30% THRU WEEK 8 MAXIMUM CHANGE > n/N (%) 3/10 (30%) 8/10 (80%) 30% THRU WEEK 12

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. 

We claim:
 1. A method for treating an HLV-infected patient consisting of administering a combination of one protease inhibitor selected from the group consisting of indinavir, nelfinavir, ritonavir and saquinavir, at least one additional antiretroviral agent that is selected from the group consisting of didanosine, lamivudine, stavudine, dalcitabine and a non-nucleoside reverse transcriptase inhibitor, and human recombinant GM-CSF, wherein said combination is administered for a time sufficient to induce an increase of at least 30% in the patient's CD4⁺ T-lymphocyte count, and further wherein said patient has received indinavir, nelfinavir, ritonavir or saquinavir for a minimum of eight weeks prior to receiving a first dose of GM-CSF.
 2. The method of claim 1, wherein the GM-CSF is administered by subcutaneous injection at a dose of 100-250 μg two times per week or three times per week.
 3. A method for treating an HIV-infected patient, comprising concurrent administration of human GM-CSF and one protease inhibitor that is selected from the group consisting of indinavir, nelfinavir, ritonavir and saquinavir, wherein said patient has received said protease inhibitor for a minimum of eight weeks prior to receiving a first dose of GM-CSF, and further wherein the QM-CSF is administered for a time sufficient to induce an increase of at least 30% in the patient's CD4⁺ T-lymphocyte count.
 4. A method according to claim 3, wherein the GM-CSF is human recombinant (GM-CSF.
 5. A method according to claim 4, wherein the human recombinant GM-CSF consists of a single 127 amino acid glycoprotein that differs from endogenous human GM-CSF by a leucine instead of a proline at position
 23. 6. The method of claim 3, wherein the (GM-CSF is administered by subcutaneous injection at a dose of 100-250 μg two times per week or three times per week. 